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DNA replication or DNA synthesis, it is the process of copying a double-stranded DNA strand. Since DNA strands are antiparallel and complementary, each strand can serve as a template for the reproduction of the opposite strand. The template strand is preserved as a whole piece and the new strand is assembled from nucleotide triphosphates. This process is called semiconservative replication. Ideally, the two resulting strands are identical, although in reality there are always errors, though proofreading and error-checking mechanisms exist to ensure a very high level of fidelity.
In a cell, this step is obligatory prior to cell division. Prokaryotes persistently replicate their DNA and creating a whole, new chromosome is a limiting step in cell division. In eukaryotes, timings are highly regulated and this occurs during the S phase of the cell cycle, preceding mitosis or meiosis I. The process may also be performed in vitro using reconstituted or completely artificial components, in a process known as PCR.
DNA may be synthesized artificially, but this process is not fundamentally a replicative process, and only produces one strand of DNA.
Contents |
Introduction
DNA replication or DNA synthesis, it is the process of copying a double-stranded DNA strand. Since DNA strands are antiparallel and complementary, each strand can serve as a template for the reproduction of the opposite strand. The template strand is preserved as a whole piece and the new strand is assembled from nucleotide triphosphates. This process is called semiconservative replication. Ideally, the two resulting strands are identical, although in reality there are always errors, though proofreading and error-checking mechanisms exist to ensure a very high level of fidelity.
In a cell, this step is obligatory prior to cell division. Prokaryotes persistently replicate their DNA and creating a whole, new chromosome is a limiting step in cell division.The large size of eukaryotic chromosomes and the limits of nucleotide incorporation during DNA synthesis, make it necessary for multiple origins of replication to exist in order to complete replication in a reasonable period of time. However, it is clear that at a replication origin the strands of DNA must dissociate and unwind in order to allow access to DNA polymerase.
The process may also be performed in vitro using reconstituted or completely artificial components, in a process known as PCR. DNA may be synthesized artificially, but this process is not fundamentally a replicative process, and only produces one strand of DNA.
3 Steps for DNA polymerization
This is a description of DNA polymerization using an enzyme. This is not the synthetic, purely chemical, laboratory method of artificially synthesizing oligos in a laboratory or oligo factory. Artificially synthesized oligos are a key aspect of the Polymerase Chain Reaction (PCR).
Initiation
In the initiation step, several key factors are recruited to an origin of replication. This origin of replication is unwound (i.e., the two strands are pulled apart at that site) and the partially unwound strands form a "replication bubble", with one replication fork on either end. Each group of enzymes at the replication fork moves away from the origin, unwinding and replicating the original DNA strands as they proceed. Primers mark the individual sequences and the starting points to be replicated.
The factors involved are collectively called the pre-replication complex. It consists of the following:
- A topoisomerase, which introduces negative supercoils into the DNA in order to minimize tortional strain induced by the unwinding of the DNA by helicase ahead of the replicational complex. The topoisomerase reversibly breaks the DNA strand, allowing the DNA to swivel, preventing the DNA from knotting up.
- A helicase, which unwinds and splits the DNA ahead of the fork. Thereafter, single-strand binding proteins (SSB) swiftly bind to the separated DNA, preventing the strands from reuniting.
- A primase (DnaG or RNA polymerase in prokaryotes, DNA polymerase α in eukaryotes), which generates an RNA primer to be used in DNA replication.
- A
DNA holoenzyme, which in reality is a complex of
proteins that together perform the "actual" replication,
i.e., the polymerization of nucleotides complementary to
the template strand.
- DNA polymerase III in prokaryotes
DNA polymerase δ and DNA pol ε in eukaryotes.
- DNA polymerase III in prokaryotes
Elongation
5' _ _ _ _ _ _ _ _ _ _ _3' <--DNA template Primer-DNA-Primer-DNA <--Okazaki fragments 3' _ _ _ _ _ _ _ _ _ _ _5' <--complimentary DNA strand
At the beginning of elongation, an enzyme called DNA polymerase binds to the DNA and synthesizes DNA from the RNA primer, which indicates the starting point for the elongation. DNA polymerases can only synthesize the new DNA in the 5’ to 3’ direction. Because of this these enzymes can only travel on one side of the original strand without any interruption. This new strand, which proceeds from 5’ to 3’, is the leading strand. The other new strand, which procedes from 3’ to 5’, is the lagging strand.
In prokaryotes RNA primers are removed by DNA polymerase I, which also synthesizes DNA in their place, in eukaryotes they are removed by RNase H or FER1 and replaced with DNA by DNA polymerase δ or ε.
Since DNA synthesis only occurs in the 5' to 3' direction, the DNA of the lagging strand is replicated in pieces known as Okazaki Fragments. Each time DNA polymerase reaches the 5' end of the RNA primer for the next Okazaki fragment, it dissociates and reassociates at the 3' end of the primer. Another enzyme, DNA ligase, is necessary to connect the Okazaki fragments.
In prokaryotes, leading strand and lagging strand synthesis are coupled by the action of the DNA polymerase III holoenzyme. One complex replicates the leading and lagging strands simultaneously. During this stage helicase continues to unwind the DNA into two single strands while topoisomerases relieves the supercoiling caused by this.
Both prokaryotes and eukaryotes have DNA polymerases with proof-reading and 3' exonuclease activities. These functions increase the fidelity of replication. Prokaryotes tend to have fewer or weaker proof-reading mechanisms due to the nature of their natural selection of their gene pools.
Termination
Termination occurs when DNA replication forks meet one another or run to the end of a linear DNA molecule. Also, termination may occur when a replication fork is deliberately stopped by a special protein, called a replication terminator protein, that binds to specific sites on a DNA molecule.
When the polymerase reaches the end of a linear DNA molecule, there is a potential problem due to the antiparallel structure of DNA. Because an RNA primer must be regularly laid down on the lagging strand, the last section of the lagging-strand DNA cannot be replicated because there is no DNA template for the primer to be synthesized on. To solve this problem, the ends of most chromosomes consist of noncoding DNA that contains repeat sequences. The end of a linear chromosome is called the telomere. Cells can endure the shortening of the chromosome at the telomere to a degree, since it's necessary for chromosome stability. Many cells use an enzyme called telomerase that adds the repeat units to the end of the chromosome so the ends do not become too short after multiple rounds of DNA replication. Many simple, single-celled organisms overcome the whole problem by having circular chromosomes.
Before the DNA replication is finally complete, enzymes are used to proofread the sequences to make sure the nucleotides are paired up correctly in a process called DNA repair. If mistake or damage occurs, enzymes such as a nuclease will remove the incorrect DNA. DNA polymerase will then fill in the gap.
Equation
A chemical equation can be written that represents the process:
(DNA)n + dNTP → (DNA)n+1 + PPi
Nucleotides (dNTP) used by DNA replications contain three phosphates attached to the sugar, like ATP and are named accordingly CTP, TTP, and GTP. However, in contrast to most other processes of the cell in which only one phosphate group (Pi), the last two phosphate groups (PPi) (pyrophosphate group) are detached.
Organization of multiple replication sites
A diploid human cell contains 6 billion nucleotide pairs (arrayed in 46 linear chromosomes) that are copied at about 50 base pairs per second by each replication fork. Yet in a typical cell the entire replication process takes only about 8 hours. This is because there are many replication origin sites on a eukaryotic chromosome. Therefore, replication can begin at some origins earlier than at others. As replication nears completion, "bubbles" of newly replicated DNA meet and fuse, forming two new molecules.
There must be some form of regulation and organization of these multiple replication sites to prevent conflict. To date, two replication control mechanisms have been identified: one positive and one negative. For DNA to be replicated, each replication origin site must be bound by a set of proteins called the origin recognition complex. These remain attached to the DNA throughout the replication process. Specific accessory proteins, called licensing factors, must also be present for initiation of replication. Destruction of these proteins after initiation of replication prevents further replication cycles from occurring. This is because licensing factors are only produced when the nuclear membrane of a cell breaks down during mitosis.
See also
- Immortal DNA strand hypothesis
External links
- DNA Workshop
- Breakfast of Champions Does Replication - a creative primer on the process from the Science Creative Quarterly
- Animation of DNA Replication (Flash Animation)
- Basic Polymerase Chain Reaction Protocol
- Animated Biology
- This article contains material from the Science Primer published by the NCBI, which, as a US government publication, is in the public domain
